Methods of inhibiting bacterial sialidase

ABSTRACT

A method of inhibiting bacterial sialidase comprising administering to a subject an inhibiting effective amount of a compound of formula I: ##STR1## wherein A is CO 2  H, PO 2  H, or SO 2  H; B is N; R 1  and R 2  are H; R 3  and R 4  are, independently, H, OH, NO 2 , guanidino, or alkyl or alkenyl of from 1 to 3 carbons where the alkyl or alkenyl is unsubstituted or is substituted, independently, with one or more of OH, NH2, or halide; R 5  is H; and R 6  is COCH 3 , or COCI 3  ; or an analog, pharmaceutically acceptable salt, derivative, or mixture thereof. A method of preventing a bacterial or trypanosomal infection using the compounds of formula I. A method of treating a bacterial or trypanosomal infection using the compounds of formula I. A pharmaceutical composition comprising a pharmaceutically acceptable carrier admixed with an inhibiting effective amount of a compound of formula I. A method of making a pharmaceutical composition, comprising admixing a pharmaceutically acceptable carrier with an inhibiting effective amount of a compound of formula I.

GOVERNMENT INTEREST

This application has been supported by two grants from the United StatesNational Institutes of Health: R01 AI26718 to Dr. Gillian Air and U01AI31888 to Dr. Ming Luo and a grant from the National Aeronautics andSpace Administration: NAGW-813 to Dr. Larry Delucas.

This application is a continuation-in-part of U.S. Ser. No. 08/227,549,filed Apr. 14, 1994, now U.S. Pat. No. 5,453,533, the contents of whichare hereby incorporated fully by this reference.

BACKGROUND

1. Field of the Invention

This invention relates to methods of and pharmaceutical compositions forinhibiting bacterial sialidase and pharmaceutical compositions thereof.In particular, this invention provides novel methods of inhibitingbacterial sialidase, modes of administration of the compounds used inthe methods, and pharmaceutical compositions for those methods.

2. Background of the Invention

Sialidases (acylneuraminyl hydrolases, EC 3.2.1.18), also known asneuramninidases, are enzymes which cleave the α-ketosidic bond between aterminal sialic acid residue and an aglycon moiety. The aglycon isusually the penultimate sugar residue of a glyco conjugate orglycoprotein carbohydrate chain. The first sialidase was purified andcharacterized from the influenza virus and the bacteria Vibrio cholerae[Gottschalk, A. (1957). Neuraminidase: The Specific Enzyme of InfluenzaVirus and Vibrio cholerae. Biochim Biophys Acta., 23, pp. 645-646].Today, sialidases specific for varying ketosidic linkages have beenidentified in viruses, bacteria, parasites, and mammals. They play acritical role in viral, bacterial, and protozoa biology by mediatingmetabolism, adherence, and infection, and are important regulators ofalternate complement pathway activation, red blood cell destruction,cell growth, cell adhesion, and tumor metastasis in mammalian systems.

Therefore, the development of sialidase inhibitors could lead to abetter understanding of these mechanisms. Also, given the wideprevalence and important role of sialidases in microbial infection, itis highly desirable to develop sialidase inhibitors to be used asanti-bacterial and anti-trypanosomal agents.

Though sialidases have long been identified in bacteria, the last twentyyears have seen an explosion of bacterial sialidases purified andcharacterized due to the advance of molecular biological techniques. Theexplosion has also shed light on sialidase's role in bacterialmetabolism, adherence, infection, and pathogenicity. Except for theactive site, the bacterial sialidases do not exhibit an amino acidsequence similarity to the viral sialidases. Another characteristic ofbacterial sialidases is the presence of non-sialidase related domains inthe protein. These domains have other activities or functions which arebeneficial to the bacteria. Many bacterial sialidases are membraneanchored, like the viral sialidases, while others are excretedextracellularly by the bacterium. Bacterial sialidases fall into twofurther subgroups based upon divalent metal requirements. The sialidasesubgroup that requires a metal ion is represented by the Vibrio choleraesialidase. The subgroup that does not require a metal ion for activityis represented by several bacterial sialidases, such as Clostridiumperfringens, Clostridium sordelli, Micromonospora viridifaciens, andSalmonella typhimurium among others. In addition to the high degree ofsequence homology within the subgroup, the non-metal requiringsialidases also show a large amount of similarity to the N-terminaltrans-sialidase domain of the trypanosomal trans-sialidase enzyme. Thecrystal structure for Salmonella typhimurium sialidase has been solved[Crennell, S. J., Garman, E. F., Laver, W. G., Vimr, E. R & Taylor, G.L. (1993), The crystal structure of a bacterial sialidase (fromSalmonella typhimurium LT2) shows the same fold as an influenza virusneuraminidase. Proc Nat Acad of Sci USA., 90, pp. 9852-6].

SUMMARY OF THE INVENTION

The present invention provides a method of inhibiting bacterialsialidase comprising administering to a subject an inhibiting effectiveamount of a compound of formula I: ##STR2## wherein A is CO₂ H, PO₂ H,or SO₂ H; B is N; R₁ and R₂ are H; R₃ and R₄ are, independently, H, OH,NO₂, guanidino, or alkyl or alkenyl of from 1 to 3 carbons where thealkyl or alkenyl is unsubstituted or is substituted, independently, withone or more of OH, NH2, or halide; R₅ is H; and R₆ is COCH₃, or COCI₃ ;or an analog, pharmaceutically acceptable salt, derivative, or mixturethereof

The present invention also provides a method of treating a bacterial ortrypanosomal infection, comprising administering to a subject apreventative effective amount a compound of formula I wherein A is CO₂H, PO₂ H, or SO₂ H; B is N; R₁ and R₂ are H; R₃ and R₄ are,independently, H, OH, NO₂, guanidino, or alkyl or alkenyl of from 1 to 3carbons where the alkyl or alkenyl is unsubstituted or is substituted,independently, with one or more of OH, NH2, or halide; R₅ is H; and R₆is COCH₃ or COCI₃ ; an analog, pharmaceutically acceptable salt,derivative, or mixture thereof

In another embodiment, the present invention provides a method ofpreventing a bacterial or trypanosomal infection, comprisingadministering to a subject a preventative effective amount a compound offormula I wherein A is CO₂ H, PO₂ H, or SO₂ H; B is N; R₁ and R₂ are H;R₃ and R₄ are, independently, H, OH, NO₂, guanidino, or alkyl or alkenylof from 1 to 3 carbons where the alkyl or alkenyl is unsubstituted or issubstituted, independently, with one or more of OH NH2, or halide; R₅ isH; and R₆ is COCH₃, or COCI₃ ; or an analog, pharmaceutically acceptablesalt, derivative, or mixture thereof.

In another embodiment, the present invention provides a pharmaceuticalcomposition for inhibiting bacterial sialidase, comprising apharmaceutically acceptable carrier admixed with an inhibiting effectiveamount of a compound of formula I wherein A is CO₂ H, PO₂ H, or SO₂ H; Bis N; R₁ and R₂ are H; R₃ and R₄ are, independently, H, OH, NO₂,guanidino, or alkyl or alkenyl of from 1 to 3 carbons where the alkyl oralkenyl is unsubstituted or is substituted, independently, with one ormore of OH, NH2, or halide; R₅ is H; and R₆ is COCH₃, or COCI₃ ; ananalog, pharmaceutically acceptable salt, derivative, or mixture thereof

In another embodiment, the present invention provides a method of makinga pharmaceutical composition for inhibiting bacterial sialidase,comprising admixing a pharmaceutically acceptable carrier with aninhibiting effective amount of a compound of formula I wherein A is CO₂H, PO₂ H, or SO₂ H, B is N; R₁ and R₂ are H; R₃ and R₄ are,independently, H, OH, NO₂, guanidino, or alkyl or alkenyl of from 1 to 3carbons where the alkyl or alkenyl is unsubstituted or is substituted,independently, with one or more of OH, NH2, or halide; R₅ is H; and R₆is COCH₃, or COCI₃ ; an analog, pharmaceutically acceptable salt,derivative, or mixture thereof.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of the inhibitory activity and the chemicalstructures of Neu5Ac2en, HNBA and GBA.

FIG. 2a shows a stereopair view of energy minimized Neu5Ac2en in thesialidase binding site.

FIG. 3a shows a stereopair view of energy minimized HNBA in thesialidase binding site. FIG. 3b shows a stereopair view of energyminimized GBA in the sialidase binding site.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of inhibiting bacterialsialidase comprising administering to a subject an inhibiting effectiveamount of a compound of formula I: ##STR3## wherein A is CO₂ H, PO₂ H,or SO₂ H; B is N; R₁ and R₂ are H; R₃ and R₄ are, independently, H, OH,NO₂, guanidino, or allyl or alkenyl of from 1 to 3 carbons where thealkyl or alkenyl is unsubstituted or is substituted, independently, withone or more of OH, NH2, or halide; R₅ is H; and R₆ is COCH₃, or COCI₃ ;or an analog, pharmaceutically acceptable salt, derivative, or mixturethereof In a preferred embodiment, A is CO₂ H; R₃ and R₄ are,independently, H, OH, NO₂, or guanidino; R₅ is H; and R₆ is COCH₃, or ananalog, pharmaceutically acceptable salt, derivative, or mixture thereofIn yet another preferred embodiment, one of R₃ and R₄ is OH the other isNO₂. In a further preferred embodiment, R₃ and R₄ is H the other isguanidino. In yet another preferred embodiment, the administering stepcomprises topical administration.

In another embodiment, the present invention provides a method oftreating a bacterial or trypanosomal infection, comprising administeringto a subject a preventative effective amount a compound of formula I:##STR4## wherein A is CO₂ H, PO₂ H, or SO₂ H; B is N; R₁ and R₂ are H;R₃ and R₄ are, independently, H, OH, NO₂, guanidino, or alkyl or alkenylof from 1 to 3 carbons where the alkyl or alkenyl is unsubstituted or issubstituted, independently, with one or more of OH, NH2, or halide; R₅is H; and R₆ is COCH₃, or COCI₃ ; or an analog, pharmaceuticallyacceptable salt, derivative, or mixture thereof In a preferredembodiment, the present invention provides this method A is CO₂ H; R₃and R₄ are, independently, H, OH, NO₂, or guanidino; R₅ is H; and R₆ isCOCH₃, or an analog, pharmaceutically acceptable salt, derivative, ormixture thereof. In yet another preferred embodiment, the administeringstep comprises topical administration.

In yet another embodiment, the present invention provides a method ofpreventing a bacterial or trypanosomal infection, comprisingadministering to a subject a preventative effective amount a compound offormula I: ##STR5## wherein A is CO₂ H, PO₂ H, or SO₂ H; B is N; R₁ andR₂ are H; R₃ and R₄ are, independently, H, OH, NO₂, guanidino, or alkylor alkenyl of from 1 to 3 carbons where the alkyl or alkenyl isunsubstituted or is substituted, independently, with one or more of OH,NH2, or halide; R₅ is H; and R₆ is COCH₃, or COCI₃ ; or an analog,pharmaceutically acceptable salt, derivative, or mixture thereof In apreferred embodiment, A is CO₂ H; R₃ and R₄ are, independently, H, OH,NO₂, or guanidino; R₅ is H; and R₆ is COCH₃, or an analog,pharmaceutically acceptable salt, derivative, or mixture thereof In yetanother preferred embodiment, the administering step comprises topicaladministration.

In another embodiment, the present invention provides, a pharmaceuticalcomposition for inhibiting bacterial sialidase, comprising apharmaceutically acceptable carrier admixed with an inhibiting effectiveamount of a compound of formula I: ##STR6## wherein A is CO₂ H, PO₂ H,or SO₂ H; B is N; R₁ and R₂ are H; R₃ and R₄ are, independently, H, OH,NO₂, guanidino, or alkyl or alkenyl of from 1 to 3 carbons where thealkyl or alkenyl is unsubstituted or is substituted, independently, withone or more of OH, NH2, or halide; R₅ is H; and R₅ is COCH₃, or COCI₃ ;or an analog, pharmaceutically acceptable salt, derivative, or mixturethereof In a preferred embodiment, A is CO₂ H; R₃ and R₄ are,independently, H, OH, NO₂, or guanidino; R₅ is H; and R₆ is COCH₃, or ananalog, pharmaceutically acceptable salt, derivative, or mixture thereof

In a further preferred embodiment, the present invention provides amethod of making a pharmaceutical composition for inhibiting bacterialsialidase, comprising admixing a pharmaceutically acceptable carrierwith an inhibiting effective amount of a compound of formula I: ##STR7##wherein A is CO₂ H, PO₂ H, or SO₂ H; B is N; R₁ and R₂ are H; R₃ and R₄are, independently, H, OH, NO₂, guanidino, or alkyl or alkenyl of from 1to 3 carbons where the alkyl or alkenyl is unsubstituted or issubstituted, independently, with one or more of OH, NH2, or halide; R₅is H; and R₆ is COCH₃, or COCI₃ ; or an analog, pharmaceuticallyacceptable salt, derivative, or mixture thereof In a preferredembodiment, A is CO₂ H; R₃ and R₄ are, independently, H, OH, NO₂, orguanidino; R₅ is H; and R₆ is COCH₃, or an analog, pharmaceuticallyacceptable salt, derivative, or mixture thereof.

A. Design of Benzenoid Inhibitors

2-Deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) is a known goodinhibitor (K_(i) =4×10⁻⁶ M) for influenza A neuramidase [N. R. Taylorand M. von Itzstein, "Molecular Modeling Studies on Ligand Binding toSialidase from Influenza Virus and the Mechanism of Catalysis," J Med.Chem., 37, 616-624 (1994)]. Evaluation of the interactions between DANAand the sialic acid binding site on neuraminidase reveal that, among theside chains found on the ring of DANA, the carboxylate most stronglyassociates with the binding site via 4 interactions to three differentarginine residues (N2 numbering: Arg 118, 292, and 371). Additionally,the N-acetyl group is the only substituent which occupies a hydrophobicpocket (interactions of methyl with Ile 222 and Trp 178, along withother interactions of the amide carbonyl oxygen with Arg 152 and theamide nitrogen with an ordered water).

For the DANA-neuraminidase complex, the plane of the N-acetyl amide isoriented perpendicular to the plane of, and bisects the O--C═O bond of,the carboxylate. This produces an amide N to Cl distance of 5.51 Å, andan amide O to Cl distance of 6.20 Å.

All known good inhibitors of influenza neuraminidase are carbohydratederivatives containing the pyran ring, and most are structurally similarto DANA. As drugs, these compounds suffer from potential problemsincluding lack of oral activity, metabolic lability, complicatedstereochemistry, difficulty in synthesis and excessive conformationalflexibility. The present invention, however, describes simplernon-carbohydrate compounds containing the carboxylate and the N-acetylgroups, or alternative groups that can undergo similar interactions withthe binding site, attached to a cyclic backbone "spacer". The cyclicspacer correctly orients the general groups or alternative side chaingroupings to provide new classes of neuraminidase inhibitors for furtherelaboration.

The benzene ring spacer has inherently simple stereochemistry (i.e, nochiral carbons occur at side chain to ring branches), relativeconformational rigidity (the predicted positioning of side chainfunctionality is simplified), relative ease of synthesis, and prospectsfor oral activity and good metabolic disposition (many useful drugs arebased upon benzene).

B. Definitions

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

The term "alkyl" as used herein refers to a branched or unbranchedsaturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, and the like.Preferred alkyl groups herein contain from 1 to 6, or more preferably 1to 4, carbon atoms. The term "lower alky" intends an alkyl group of fromone to six carbon atoms, preferably from one to four carbon atoms.

The term "alkoxy" as used herein intends an alkyl group bound through asingle, terminal ether linkage; that is, an "alkoxy" group may bedefined as --OR where R is alkyl as defined above. A "lower alkoxy"group intends an alkoxy group containing from one to six, morepreferably from one to four, carbon atoms. Likewise, the terms"alkenoxy" and "alkynoxy" as used herein intend an alkenyl or alkynylgroup bound through a single, terminal ether linkage; that is, an"alkenoxy" or "alkynoxy" group may be defined as --OR where R is alkenylor alkynyl as defined above.

The term "alkenyl" as used herein intends a mono-unsaturated orpoly-unsaturated hydrocarbon group of 2 to 24 carbon atoms. Preferredgroups within this class contain 2 to 12 carbon atoms. Likewise, theterm "alkynyl" as used herein intends a hydrocarbon group of 2 to 24carbon atoms containing at least one triple bond. Preferred groupswithin this class contain 2 to 12 carbon atoms.

The term "halide" is used to refer to any halogen including, F, Cl, I,or Br.

The terms "cyclic" and "heterocyclic" refer to rings where,respectively, none or one or more of the carbon atoms have beenreplaced. For instance, for a "heterocyclic" ring, a carbon in the ringmay be preferably substituted with N, O, or S. Such atoms which aresubstituted are herein called "heteroatoms." One of skill in the artwould recognize that other suitable heteroatoms exist.

The term "core ring" is used to refer to the base six-membered ringdepicted in general structure I.

The terms "saturation" and "unsaturation" are used to describe whether,between a particular pair of atoms, a single or double bond exists.Single bonds are termed "saturations" and double bonds are termed"unsaturations." One of skill in the art would recognize that triplebonds could also constitute "unsaturations". Furthermore, the terms"saturated" and "partially unsaturated" and "fully unsaturated" are usedto refer to the presence or lack of unsaturations in a particular ring.For instance, cyclohexane would be considered a "saturated" compound. Onthe other hand cyclohexene would be "partially unsaturated" due to thepresence of one unsaturation. Finally, benzene is "fully unsaturated"due to the presence of the maximum, three, unsaturations.

The terms "alkanol", "alkenol" and "alkynor", as used herein, refer tothe alcohol versions of respective alkanes, alkenes and alkynes. Thealcohols may contain one or more OH moieties. Furthermore, the alcoholsmay be branched or straight and the OH moieties may be present at theterminal carbons or elsewhere along the carbon chain. More than one OHgroup may be subsituted at any particular carbon. Examples of "alkanols"are methanol, ethanol, CH₃ CH(OH)₂, etc. Examples of alkenols includeCH₂ CHOH, CH₃ CH₂ CHOH, etc. An example of an alkynol is CH₃ CH₂ CCOH.As used in the claims, a substitution of an alkanol implies that one ofthe hydrogens is removed at the linking atom and that atom is bonded tothe entity having the substitution. The same interpretation applies toall other moieties described in this specification where the contextrequires such interpretation.

As used herein, "subject" is intended to cover humans, mammals and otheranimals which are susceptible to bacteria in any fashion.

By the term "effective amount" of a compound as provided herein is meanta nontoxic but sufficient amount of the compound to provide the desiredutility. For instance, for inhibition of sialidase, the effective amountis the amount which provides clinically meaningful inhibition ofsialidase in a subject. As will be pointed out below, the exact amountrequired will vary from subject to subject, depending on the species,age, and general condition of the subject, the severity of the conditionor disease that is being treated, the particular compound used, its modeof administration, and the like. Thus, it is not possible to specify anexact "effective amount." However, an appropriate effective amount maybe determined by one of ordinary skill in the art using only routineexperimentation.

By "pharmaceutically acceptable" is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected bicyclic compoundwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

Pharmaceutically acceptable salts are prepared by treating the free acidwith an appropriate amount of a pharmaceutically acceptable base.Representative pharmaceutically acceptable bases are ammonium hydroxide,sodium hydroxide, potassium hydroxide, lithium hydroxide, calciumhydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide,copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine,arginine, histidine, and the like. The reaction is conducted in water,alone or in combination with an inert, water-miscible organic solvent,at a temperature of from about 0°0 C. to about 100° C., preferably atroom temperature. The molar ratio of compounds of general structure I tobase used are chosen to provide the ratio desired for any particularsalts. For preparing, for example, the ammonium salts of the free acidstarting material, the starting material can be treated withapproximately one equivalent of pharmaceutically acceptable base toyield a neutral salt. When calcium salts are prepared, approximatelyone-half a molar equivalent of base is used to yield a neutral salt,while for aluminum salts, approximately one-third a molar equivalent ofbase will be used. "Salt" is further defined elsewhere herein.

As used herein, and without limitation, the term "analog" is used torefer to any compound which has structural similarity to the compoundsof the invention and would be expected, by one skilled in the art, toexhibit the same or similar utility as the claimed compounds.

As used herein, and without limitation, the term "derivative" is used torefer to any compound which has a structure derived from the structureof the compounds of the present invention and whose structure issufficiently similar to those disclosed herein and based upon thatsimilarity, would be expected, by one skilled in the art, to exhibit thesame or similar activities and utilities as the claimed compounds.

C. Synthesis of Benzenoid Inhibitors

Compounds with General Structure I and their pharmaceutically acceptablesalts and derivatives, may be prepared using any of several methodsknown in the art for the synthesis of substituted benzenoid compoundscontaining analogous structures. ##STR8##

To illustrate, potential synthetic approaches for selected examples fromGeneral Structure I are summarized in the following two reaction schemesand are representative of the types of procedures to be employed. Table1 lists some of the compounds successfully synthesized to date.

                  TABLE 1                                                         ______________________________________                                        1  STR9##                                                                        -                                                                              Compound   R.sub.1 R.sub.2                                                                              R.sub.3                                                                             R.sub.4                                   ______________________________________                                        101        H       H        H     OH                                            102     H      H      H              OAc                                      103    NO.sub.2    NO.sub.2     H              OH                             104     H      H     NO.sub.2             OAc                                 105 HNBA   H      H     NO.sub.2             OH                               113 GBA   H      H      H           NHC(NH)NH.sub.2                         ______________________________________                                    

Table 2 proposes constructing a basic skeleton via formylation ortho tothe acetylamino group. This could be accomplished using Friedel-Craftsalkylation with dichloromethyl methyl ether, which has been shown to bea general method for the formylation of numerous substituted benzenes[A. Rieche, H. Gross, and E. Hoft, "Synthesis of Aromatic Aldehydes withDichloromethyl-Alkyl Ethers," Chem. Ber., 93, 88-94 (1960)]. Asillustrated here, the o-formylation of 14 will provide target 24 (viaintermediate 23), and the o-formylation of 25-27 will provide precursors28-30 for the further elaboration to additional targets.

                  TABLE 2                                                         ______________________________________                                        Scheme for Synthesizing Benzenoid Candidate Inhibitors                        ______________________________________                                        2  STR10##                                                                      3  STR11##                                                                        4  STR12##                                                                        PATHWAY A:                                                                    5  STR13##                                                                      6 #STR14##                                                        ______________________________________                                    

This key o-formylation step occurs early in the proposed syntheses, andif unexpected difficulties are encountered, several alternatives arepossible. These include: (a) Other o-formylation methods could beemployed, such as the o-formylation of anilines via the rearrangement ofazasulfonium salts [P. G. Gassman and H. R. Drewes, "The OrthoFunctionalization of Aromatic Amines. Benzylation, Formylation, andVinylation of Anilines," J Am. Chem. Soc., 100, 7600-7610 (1978)], whichhas been used for the synthesis of substitutedo-acetylamino-benzaldehydes. (b) Since the acetylamino group is a good"directed metalation group" [V. Snieckus, "Directed Ortho Metalation.Tertiary Amide and O-Carbamate Directors in Synthetic Strategies forPolysubstituted Aromatics," Chem. Rev., 90, 879-933 (1990)], it ispossible to regioselectively o-lithiate suitable derivatives of 14 and25-27. Reaction with appropriate electrophiles (epoxides, alkyl halides,aldehydes, etc.) would then provide an entry into desired targets. (c)The o-iodination of protected 14 and 25-27 could be employed inanticipation of a Heck-type coupling reaction [Y. Hatanaka, Y. Ebina,and T. Hiyama, "γ-Selective Cross-Coupling Reaction ofAllyltrifluorosilanes: A New Approach to Regiochemnical Control inAllylic Systems," J Am. Chem. Soc., 113, 7075-7076 (1991); K. Nilssonand A. Hallberg, "Synthesis of 1-Propyl-3-(3-Hydroxyphenyl)piperidine byRegiocontrolled Palladium-Catalyzed Acylation," J Org. Chem., 57,4015-4017 (1992)] to introduce o-substituents. Pathway A describes theproposed elaboration of precursors 28 and 29 to final products. Wittigolefination of the benzaldehydes will provide 31 and 32, andhydroboration-oxidation using thexylchloroborane [H. C. Brown, J. A.Sikorski, S. U. Kulkamni, and H. D. Lee, "Thexylchloroborane-MethylSulfide. A Selective Monohydroborating Agent with ExceptionalRegioselectivity," J Org. Chem., 45, 4540-4542 (1980)] will providehydroxyethyl derivatives 33 and 34. Basic hydrolysis of the esters thenprovides targets 35 and 36.

A similar procedure is proposed in Pathway B (Table 3) for the formationof additional targets. In this procedure 28-30 undergo conversion toiodomethyl derivatives 37-39, which are coupled with two differentlithium dialkyl cuprates [G. Posner, "Substitution Reactions UsingOrganocopper Reagents," Org. React., 22, 253-400 (1975)] to provide40-44. Glycol formation using N-methylmorpholine-N-oxide and catalyticOsO₄ [N. Iwasaw, T. Kato, and K. Narasaka, "A Convenient Method forDihydroxylation of Olefins by the Combined Use of Osmium Tetroxide andDihydroxyphenylborane," Chem. Lett., 1721-1724 (1988)], or hydroborationas in Pathway A, followed by basic hydrolysis, then provides the finalproducts 45-53

                                      TABLE 3                                     __________________________________________________________________________    Scheme for Synthesizing Benzenoid Candidate Inhibitors                        __________________________________________________________________________    PATHWAY B:                                                                    7  STR15##                                                                    8 #STR16##                                                                    __________________________________________________________________________

The following detailed examples for methods of preparation are forillustration only, and are not intended to represent a limitation of theinvention. The structures of the compounds whose preparations aredescribed below are summarized in Table 1. In all cases syntheticintermediates and products were found to be pure according to standardsknown to those skilled in the art (such as thin layer chromatography,melting or boiling points, gas chromatography, ion exchangechromatography, and/or high pressure liquid chromatography, elementalanalysis, and spectroscopic methods). Furthermore, structures werecharacterized and fully assigned by spectroscopic methods consideredstandard practices by those skilled in the art (such as infrared,ultraviolet, and mass spectroscopies, ¹ H and ¹³ C nuclear magneticresonance spectroscopy, and/or x-ray crystallography). Selected spectraldata are described for intermediates and products.

EXAMPLE 1

The preparations of 4-(acetylanino)-3-hydroxybenzoic acid (101) and4-(acetylamino)-3-acetoxybenzoic acid (102). The overall reaction schemeis shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Reaction Paths and the Chemical Structures of                                   Compounds 13, 102 and 101                                                   ______________________________________                                        9  STR17##                                                                      0 #STR18##                                                                  ______________________________________                                    

Preparation of 4-(acetylamino)-3-hydroxybenzoic acid (101)

A solution of 102 (100 mg, 0.42 mmol) in 0.1 N NaOH (5 mL) was stirredat room temperature for 30 minutes. Concentrated HCl was added dropwiseto adjust the mixture to pH 2, and this was extracted with ethyl acetate(2×10 mL). The extracts were dried (Na₂ SO₄), concentrated to dryness ona rotary evaporator, and the solid residue was washed out of the flaskwith dry hexane and filtered to give 101 (40 mg, 49% yield): mp 249-250°C.

¹ H NMR (D)MSO-d₆) 2.13 (s, 3 H, COCH₃), 7.37 (dd, 1 H, aromatic, J=8 &1.8 Hz), 7.44 (d, 1 H, aromatic, J=1.8 Hz), 8.01 (d, 1 H, aromatic, J=8Hz), 9.26 (s, 1 H, NH).

Preparation of 3-acetox-4-(acetylamino)benzoic acid (102)

To a stirred solution of commercially available compound 13 (0.50 g, 3.3mmol) in 2N HCl (10 mL) at 0° C. (ice bath) was added a solution ofNaOAc (5.0 g, 61 mmol) in water (25 mL). To this was added Ac₂ O (5.4 g,53 mmol). The mixture was stirred at 0° C. for 5 minutes, and it wasthen allowed to warm slowly to room temperature as the ice bath melted.After 4 hours a light brown precipitate had formed. This was filtered,washed with water (25 mL), and air-dried to provide4-(acetylamino)-3-acetoxybenzoic acid (102; 450 mg, 58% yield): mp219-221° C. (CH₃ OH/H₂ O).

¹ H NMR (DMSO-d₆) 2.17 (s, 3 H, COCH₃), 2.35 (s, 3 H, COCH₃), 7.69 (d, 1H, aromatic, J=1.8 Hz), 7.79 (dd, 1 H aromatic, J=7 & 1.8 Hz), 8.17 (d,1 H, aromatic, J=7 Hz), 9.3 (s, 1 H, NH).

EXAMPLE 2

The preparations of 4-(acetylamino)-3-hydroxy-2,6-dinitrobenzoic acid(103), 3acetoxy-4-(acetylamino)-5-nitrobenzoic acid (104),4-(acetylamino)-3-hydroxy-5-nitrobenzoic acid (105), and3-amino-4-(acetylamino)-5-hydroxybenzoic acid, hydrochloride (106). Theoverall reaction scheme is shown in Table 5.

                                      TABLE 5                                     __________________________________________________________________________    Reaction Paths and the Chemical Structures of Compounds 102, 103, 104,        105 and 106                                                                   __________________________________________________________________________    1  STR19##                                                                    2 #STR20##                                                                    __________________________________________________________________________

Preparation of 4-(acetylamino)-3-hydroxy-2,6-dinitrobenzoic acid (103)

3-Acetoxy-4-(acetylamino)benzoic acid (102; 2.00 g, 8.43 mmol) was addedgradually to a paste made from concentrated H2SO4 (8.5 mL) and potassiumnitrate (2.22 g, 22.0 mmol) at -10-0° C. The reaction was stirred at 0°C. for 30 minutes, and the syrupy mass was poured onto cracked ice.After standing for one hour a yellow solid separated. This was filtered,washed on the filter with cold water, and air-dried to give 103 (1.60 g,66.7% yield): mp 199-203° C. (ethyl acetate/hexane).

1H NMR (DMSO-d6) 2.16 (s, 3 H, NCOCH3), 8.73 (s, 1 H, aromatic), 9.72(s, 1 H, NH).

Preparation of 3-acetoxy-4-(acetylamino)-5-nitrobenzoic acid (104)

Compound 102 (1.00 g, 4.21 mmol) was suspended with stirring in amixture of Ac2O (8.33 mL, 8.99 g, 88.2 mmol) and dioxane (6.6 mL). Thiswas cooled to 0° C., and a cold solution of the nitrating mixture madefrom Ac2O (3.33 mL, 3.59 g, 35.2 mmol) and concentrated HNO3 (3.33 mL)was slowly added to the mixture containing 102. The reaction mixture wasthen warmed to 30-35° C. until the reaction was complete as evidenced byTLC. The reaction mixture was poured onto ice/water (100 mL), extractedwith EtOAc (4×50 mL), dried (NaSO4), and concentrated to dryness on arotary evaporator to give crude 104 (1.05 g, 89.9%) as an oil.Trituration with CHCl3 gave a solid: mp 196-201° C. (dioxane/hexane).

1H NMR (CD3OD) 8.42-8.33 (d, J=1.5 Hz, 1 H, aromatic), 8.05-8.12 (d,J=1.5 Hz, 1 H, aromatic), 2.35 (s, 3 H, OCOCH3), 2.13 (s, 3 H, NCOCH3).

Preparation of 4-(acetylamino)-3-hydroxy-5-nitrobenzoic acid (105)

Compound 104 (0.850 g, 3.54 mmol) was dissolved in 0.1N NaOH (80 mL),and the mixture was stirred at room temperature for 4 hours. This wasacidified with concentrated HCl (2 mL), diluted with water (20 mL), andextracted with ethyl acetate (3×60 mL). The combined extracts were dried(Na₂ SO₄) and concentrated on a rotary evaporator to give crystalline105 (0.712 g, 98.4% yield): mp 256-259° C. (methanol).

¹ H NMR (DMSO-d₆) 10.93 (s, 1H, COOH), 9.90 (s, 1H, NH), 7.79-7.69 (mn,3 H, aromatic), 2.04 (s, 3 H, NCOCH₃).

Preparation of 3-amino-4-(acetylamino)-5-hydroxybenzoic acid,hydrochloride (106)

Compound 105 (100 mg, 0.416 mmol) was dissolved in ethanol (3 mL), andPd-C (100 mg) was added to it. To this mixture was added hydrazinehydrate (55% hydrazine, 0.10 mL, 55 mg, 1.7 mmol) dropwise. The reactionmixture was heated at reflux for 1 hour. The Pd-C was filtered and theethanol was concentrated under vacuum to give the free amine of 106 as apale yellow oil (85 mg, 97% yield). The oil was dissolved in ethanol (3mL), HCl (gas) was bubbled through the solution for a few minutes, andether (10 mL) was added. No precipate formed, so the solution wasconcentrated to dryness to give 106 (100 mg) as the hydrochloride salt:mp 220° C. (dec).

¹ H NMR (D₂ O) 7.45-7.40 (m, 2 H, aromatic), 2.22 (s, 3 H, NCOCH₃).

EXAMPLE 3

The preparations of 4-(acetylamino)-3-hydroxy-5-nitrobenzoic acid (107),4-(acetylamino)-3-aminobenzoic acid (108), and4-(acetylamino)-3-guanidinobenzoic acid (113). The overall reactionscheme is shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Reaction Paths and the Chemical Structures of                                   Compounds 14, 107, 108 and 113                                              ______________________________________                                        3  STR21##                                                                      4 #STR22##                                                                  ______________________________________                                    

Preparation of 4-(acetylamino)-3-hydroxy-5-nitrobenzoic acid (107)

Commercially available compound 14 (5.00 g, 27.9 mmol) was graduallystirred into a paste prepared by adding finely pulverized potassiumnitrate (6.70 g, 66.3 mmol) to concentrated H₂ SO₄ (30 mL), and themixture was stirred at -10 to 0° C. in a salt/ice bath for one hour. Thesyrupy mass was then slowly poured onto cracked ice. After standing forone hour the yellow precipitate was filtered, washed on the filter withcold water, and air-dried to give 107 (5.27 g, 84.3% yield): mp 215-20°C (ethanol). The literature (Verma and Khan, 1978) reports mp 218-220°C.

¹ H NMR (DMSO-d6) 10.57 (s, 1 H, COOH), 8.36 (s, 1 H, aromatic), 8.2 (d,J=8.5 Hz, 1 H, aromatic), 7.82 (d, J=8.5 Hz, 1H, aromatic), 2.11 (s, 3H, COCH₃).

Preparation of 4-(acetylamino)-3-aminobenzoic acid hydrochloride (108)

To a stirred mixture of 107 (1.00 g, 4.46 mmol) and 10% Pd-C (1.0 g) inethanol (10 mL) and 5% HCl (1.2 mL) was added dropwise hydrazine hydrate(55% hydrazine, 1.0 mL, 17 mmol). The mixture was stirred at roomtemperature for 1 hour, the catalyst was filtered, and the filtrate wasconcentrated under reduced pressure. Compound 108 (0.875 g, 100%) wasobtained as a white solid residue: mp 220-223° C. (methanol/hexane). Aliterature reference (Ellis and Jones, 1974) did not report the mp.

¹ H NMR (CD₃ OD) 7.49-7.29 (m, 2 H, aromatic), 7.20-7.12 (m, 1 H,aromatic), 2.16 (s, 3 H, COCH₃).

Preparation of 4-(acetylamino)-3-guanidinobenzoic acid (113)

A mixture of 108 (100 mg, 0.43 mmol) and cyanamide (27 mg, 0.65 mmol)was mixed and heated at 85-100 C. with stirring for 15 minutes. Theliquid mass was cooled to room temperature, dissolved in hot water (0.7mL), and acidified with 1-2 drops of conc. HCl. The thick solid whichseparated was filtered and dried to give crude 113 (160 mg). This wasrecrystallized from 5% HCl to give the pure hydrochloride salt of 113(50 mg, 52%): mp 284-287 C.

¹ H NMR (TFA) 2.50 (s, 3 H, NCOCH₃), 6.50 (bs, 4 H, C(NH₂)₂ ⁺), 7.55 (d,1 H, aromatic), 8.25-8.44 (m, 3 H, aromatic & NHC(NH₂)₂ ⁺), 9.30 (bs, 1H, NHAc).

D. Structure-Based Solutions

Structure-based drug design developed out of the fact that potentialdrugs had previously been discovered only serendipitously or by the useof extensive screening assays. To improve the therapeutic properties ofexisting compounds, traditional methods such as qualitativestructure-activity relationship (QSAR) analysis have been used. However,to clearly understand the multitude of forces which affect a drug'sbiological activity (or lack thereof), the three-dimensional structureof the native target, or more preferably, the complex between the targetand the drug, must be solved. In many cases, the structure of aparticular drug-target complex can provide an immediate explanation tolong-standing and perplexing biochemical questions of function andactivity. Hence, the method of structure-based drug design, which usesthe three-dimensional structure of a selected target or target-drugcomplex to guide the design new compounds. By starting with thestructure of the target, the structure-based drug design protocolcircumvents the problems and limitations associated with traditionalmethods of drug development. New compounds that show high specificityand affinity for the target site can be developed using the chemical andgeometric structure of target site at high resolution andstructure-based design [Ealick, S. E., Babu, Y. S., Bugg, C. E., Erion,M. D., Guida, W. C., Montgomery, J. A. & Secrist III, J. A. (1991).Application of crystallographic and modeling methods in the design ofpurine nucleoside phosphorylase inhibitors. Proceedings of the NationalAcademy of Science, USA 88, 11540-44].

As recently as 1987, to develop a marketable new drug by traditionalmethods cost an estimated $231 million and required 12 years. Theenormous number of potential compounds that are chemically synthesizedcontributes to the high cost of traditional development methods. Also,traditional methods are inefficient because many synthesized compoundsare eventually rejected due to poor activity or adverse side effects. Incomparison, the time requirement and costs of developing a drug usingstructure-based design methods are much lower. In this newer approach,the candidate drugs are modeled into the three-dimensional structure ofthe target before synthesis and only sterically and chemicallycompatible structures are synthesized. The screening process furtherincreases the likelihood that the candidate compounds will bind to theactive site. The current invention targets bacterial sialidase.

E. Structure-Based Design of Anti-Bacterial and Anti-TrypanosomalCompounds

Bacterial sialidases have been implicated and correlated with severaldisease such as, inter alia, dental caries, bacterial vaginosis, middleear effusions, arteritis, acne, and acute streptococcal infection.Though many antibiotics are available to treat bacterial infections,they are often expensive or have significant side effects for thesubject. The bactericidal agents of the present invention, however, donot suffer from the expense or potential side effects. Also, basicscientific research into the role of sialidases in bacterial biology andinfection would benefit from the elucidation of bacterial-specificsialidase inhibitors.

In addition to bacteria, there are unique enzymes found only intrypanosomes which are also ideal targets for developinganti-trypanosomal therapeutics. One trypanosomal target is thecell-surface anchored trans-sialidase enzyme, which transfers a terminalsialic acid from a donor sialoglycoconjugate to a terminal β-1,4-linkedgalactose acceptor. The trans-sialidase enzyme found in the protozoaTrypanosomatidae family is believed to play a important role in severalhuman diseases, such as Chagas' disease (Trypanosoma cruzi) and Africansleeping disease (Trypanosoma gambiense and T. rhodesiense), as well as,in several animal trypanosomiasis (Trypanosoma brucei, and potentiallyT. evansi, T. congolense, and T. vivax). It is estimated that there areseveral million cases of Chagas' disease in Central and South America,as well as several millions cases of African sleeping sickness insub-Sahara Africa. Every year, several thousand new cases of Chagas'disease and African sleeping sickness occur. The rate of trypanosomiasisin the animal kingdom, which can have serious health and economicimplications, is difficult to quantify but is potentially severalmillion cases. Though previous drugs have been developed to treat thetrypanosomal infections in humans and animals, they are eitherrelatively toxic to the host or the target trypanosome strains havedeveloped a drug resistance mechanism. Inhibitors of trans-sialidasepromise to be effective anti-trypanosomal agents because thetrypanosomal trans-sialidase enzyme has been shown to be required forinfection in humans, as well as, in animals (limited data) [de Titto, E.H. & Araujo, F. G. (1987). Mechanism of cell invasion by Trypanosomacruzi: importance of sialidase activity. Acta Trop. (Basel), 44, pp.273-82; Ming, M., Chuenkova, M., Ortega-Barria, E. & Pereira, M. E.(1993). Mediation of Trypanosoma cruzi invasion by sialic acid on thehost cell and trans-sialidase on the trypanosome. Mol BiochemParasitol., 59, pp. 243-52; and Prioli, R. P., Mejia, J. S. & Pereira,M. E. (1991). On the interaction of Trypanosoma cruzi neuraminidase andhuman lipoproteins. Eur J Epidemiol., 7, pp. 344-8]. The structure-baseddesign of the present invention has led to specific drugs for thetrans-sialidase active site. Based on those structures, the drugs of thepresent invention have a reduced possibility of harming the host oreliciting a drug resistance due to mutation of the target site.

F. Salmonella typhimurium Sialidase Activity

The sialidase gene, nanH, from the enteric Gram-negative bacterium S.typhimurium has been cloned and the expressed bacterial sialidase hasbeen well characterized [Hoyer, L. L., Roggentin, P., Schauer, R. &Vimr, E. R. (1991). Purification and properties of cloned Salmonellatyphimurium LT2 sialidase with virus-typical kinetic preference forsialyl alpha 2→3 linkages. J Biochem., 110, pp. 462-7; Hoyer, L. L.,Hamilton, A. C., Steenbergen, S. M. & Vimr, E. R. (1992). Cloning,sequencing and distribution of the Salmonella typhimurium LT2 sialidasegene, nanH, provides evidence for interspecies gene transfer. MolecularMicrobiology 6(7), 873-84]. No significant differences were detected inthe expressed enzyme as compared to the wild type sialidase, except inthe wild type strain, the sialidase accounts for <1% of the totalprotein. The S. typhimurium sialidase has a 260-fold cleavage preferencefor α2→3 over α2→6 linked sialic acids. In addition, the S. typhimuriumsialidase has a high enzymatic activity for ganglioside and mucinsubstrates containing terminal sialic acids. The S. typhimuriumsialidase does not efficiently recognize α2→8 or α2-9 linked sialicacids and therefore shows little cleavage activity for colominic acid,which is a homopolymer of sialic acid, or Group C polysaccharides.

Like the influenza virus sialidase, the S. typhimurium sialidase isactive over a broad pH range of pH 5.5-7.0, but unlike viral sialidase,the bacterial sialidase does not require divalent metal ions foractivity. Using 4-methylumbelliferyl-a-D-N-acetylneuraminic acid (MUN)as the substrate, the S. typhimurium sialidase displays a K_(m)=2.5×10⁻⁴ M, and a turnover number =2,700 sec⁻¹. The dehydro analog ofsialic acid, Neu5Ac2en, inhibits S. typhimurium sialidase with a K_(i)=0.38 mM. As compared to influenza virus sialidase, high levels of thecleavage product, Neu5Ac, do not inhibit the bacterial sialidase.

G. Salmonella tphimurium Sialidase Structure

The S. typhimurium sialidase has a molecular weight of 41 kDa and apI≧9. As stated above, the three-dimensional structure of S. typhimuriumwas determined using x-ray crystallography [Crennell et al., 1993]. Thestructure was solved to 2.0 Å by the multiple isomorphic replacementmethod and refined to a crystallographic R-factor of 18.9%. Likeinfluenza virus sialidase, the S. typhimurium sialidase is folded into aleft-handed propeller motif consisting of six, four-strandedantiparallel β-sheets. The length of the β-strands and the loopsconnecting the β-strands differs markedly from the viral sialidasestructure. One disulfide bond is observed in the S. typhimuriumsialidase which links the first and second β-sheets.

H. Trypanosomal Trans-sialidase

The atomic structure of the N-terminal trans-sialidase domain of thetrypanosomal trans-sialidase protein has not been solved. But thetrypanosomal trans-sialidase N-terminal domain has a high sequencehomology to the S. typhimurium sialidase [Pereira, M. E., Mejia, J. S.,Ortega-Barria, E., Matzllevich, D. & Prioli, R. P. (1991). TheTrypanosoma cruzi neuraminidase contains sequences similar to bacterialneuraminidases, YWTD repeats of the low density lipoprotein receptor,and type III modules of fibronectin. J Exp Med., 174, pp. 179-91]. Usingthe sequence alignment of the trypanosomal trans-sialidase to thebacterial sialidase and using the S. typhimurium sialidase crystalstructure, a three-dimensional model of the trans-sialidase active sitewas constructed.

The amino acid sequence for trans-sialidase determined by Pereira et al(1991) was compared to the sialidases isolated from the bacteriaClostridium perfringens and Salmonella typhimurium. The GCG package[Program Manual for the Wisconsin Package, Version 8, September 1994,Genetics Computer Group, Madison, Wis.] of alignment programs was usedto align the three sialidase sequences. The protein database entriesused were styneur.pep (S. typhimurium) and cfsiali.pep (C. perfringens).The inclusion of other bacterial sialidase sequences in addition to theSalmonella and Clostridium sequences did not improve the overall fit ofthe of the bacterial enzymes to the trans-sialidase. The major featuresof the proposed bacterial sialidase-trypanosomal trans-sialidasesequence alignment are as follows.

First, the sequence alignment did not predict a trans-sialidase partnerfor Arg 37 in S. typhimurium sialidase sequence. This arginine is partof the arginine triad found in all influenza and bacterial sialidases todate and is required to bind the carboxylate group of the substrate,sialic acid. Second, three cysteine residues in the bacterial sialidaseare replaced by non-cysteine residues in the predicted trans-sialidasealignment. The three residues affected are Cys 103, Cys 225, and Cys 344(S. typhimurium numbering). Third, two non-cysteine residues in thebacterial sialidase are predicted to be cysteine residues in thetrypanosomal trans-sialidase. The residues in S. typhimurium whichchange to cysteines in the trans-sialidase are Lys 94 and Gly 229. Note,neither the Lys or Gly residues are conserved in the C. perfringenssialidase.

On the basis of the proposed sequence alignment, the active siteresidues of the Salmonella structure were replaced with thetrans-sialidase residues identified from the sequence alignment. Theprogram SAM in the FRODO package was used the construct thetrans-sialidase homology model. SAM builds the new residues using theoriginal residue atom positions of the S. typhimurium crystal structure.One round of limited energy minimization was applied to thetrans-sialidase homology model.

I. In Vitro Testing of Benzoic Acid Inhibitors Against BacterialSialidase

Aside from moderate inhibitory activity, carbohydrate based inhibitorsof sialidase such as Neu5Ac2en suffer as potential therapeutics due tothe unfavorable economics and difficulty of manufacturing large scaleamounts of the compounds. Benzoic acid based sialidase inhibitors arechemically easier and cheaper to synthesize.

In addition, there are numerous common synthetic routes available toselectively modify a benzene ring with different chemical functionalgroups. Furthermore, substitution of a benzene ring for the sugar ringof Neu5Ac2en does not dramatically affect inhibitory activity. Previousstudies have shown that the sugar ring of the carbohydrate-basedinhibitors does not interact directly with the protein active site andonly serves as a scaffolding to direct the placement of the inhibitorfunction groups. The similar geometry and size of a benzene ring to thecarbohydrate ring of Neu5Ac2en supports the hypothesis that a benzenering could act as a scaffolding element for inhibitor functional groupsand, therefore, benzoic acid based compounds could inhibit sialidaseactivity.

Two benzoic acid derivatives, 4-acetylamino-3-hydroxyl-5-nitro-benzoicacid (HNBA) and 4-acetylaniino-3-guanidino-benzoic acid (GBA), whichare, respectively, millimolar and micromolar inhibitors of influenzavirus sialidase, were tested for inhibitory activity against bacterialsialidase (Micromonospora viridifaciens) using a fluorescence assay andthe substrate 4-methylumbelliferyl-a-D-N-acetylneuraminic acid (MUN).FIG. 1 shows the relative inhibition activity for the compounds HNBA andGBA versus Neu5Ac2en (bacterial sialidase K₁ ×10⁶ M) determined for M.viridifaciens bacterial sialidase.

The inhibition activities were determined using a modified standardfluorometric assay employing 4-methylumbelliferyl-a-D-N-acetylneuraminicacid (MW as the substrate. Since the benzoic acid compounds have a lowsolubility in water, a 100 mM stock solution of the benzoic acidcompounds was prepared in the organic solvent dimethyl sulfoxide (DMSO).An appropriate amount of the DMSO stock solution was added to thereaction mixture which contained a final concentration of 10 mMinhibitor, 0.1 mM MUN, 50 mM NaAc (pH 6.0), 0.075 mM CaCl₂, 0.240 mMMgCl₂, 0.045 mM NaCl, and bacterial sialidase (diluted to give a linearresponse range in the fluorometer). Following addition of MUN, thesample was incubated at 37° C. for 15 minutes. The reaction was stoppedby the addition of 25 mM HEPES (pH 11.0). The amount of fluorescentproduct generated in the reaction was measured using an excitementwavelength of 365 nm and emission wavelength of 450 nm.

The inhibition activity of Neu5Ac2en was standardized against abackground control containing only water and no Neu5Ac2en. Likewise, theinhibition activity of the benzoic acid compounds was standardizedagainst a control containing only DMSO to negate the potentialinhibition effects of DMSO on bacterial sialidase. Due tostandardization, the inhibition activities of Neu5Ac2en and the benzoicacid inhibitors are reported as percent inhibition values. The percentinhibition of Neu5Ac2en serves as a positive control for inhibition bythe benzoic acid compounds.

J. Modeling of Inhibitors into Bacterial Sialidase

The compounds, HNBA and GBA, are potent inhibitors of both type A and Binfluenza virus sialidase. The IC₅₀ for HNBA inhibition of influenzavirus sialidase is approximately 10 mM, similar to the Neu5Ac2en IC₅₀for influenza virus sialidase inhibition. Based on the inhibition assayresults, HNBA and GBA also inhibited bacterial sialidase withefficiencies similar to Neu5Ac2en.

Initial modeling of the benzoic acid lead compounds into the bacterialsialidase was guided by the crystal structure of the Neu5Ac2en-bacterialsialidase complex. The placement of the benzoic acid inhibitors into thebacterial active site by superposition onto the Neu5Ac2en position didnot disturb the overall geometry of the active site, as evidenced by thelow root-mean-square deviation between the energy minimized benzoic acidand Neu5Ac2en bacterial sialidase complexes.

In the bacterial sialidase, superposition of GBA to align itsguanidinium group to the Neu5Ac2en O4 hydroxyl group, which will mimicthe 4-guanidino-Neu5Ac2en binding mode, is not possible due to stericconflicts with residues Arg 56 and Asp 100 in bacterial sialidase. Thissteric interference in the Neu5Ac2en O4 pocket also explains theinhibition selectivity of 4-guanidino-Neu5Ac2en for influenza virussialidase versus bacterial sialidases. The rotation of the GBA ringresulting from energy minimization may be due to the lack of an hydroxylgroup at the C5 position in GBA, which would bind in the Neu5Ac2en O4hydroxyl site.

K. Modeling of Inhibitor-Bacterial Sialidase Complexes

The structure of a prototypical bacterial sialidase from S. typhimuriumcomplexed with the inhibitor Neu5Ac2en by Crennell et al. was used toposition the benzoic acid inhibitors BNBA and GBA into the bacterialsialidase active site. The interactions between Neu5Ac2en and the activesite of the bacterial sialidase are shown in FIG. 2a.

To model the HNBA-bacterial sialidase complex, the C1, O3, and atoms ofHNBA were superimposed onto the C2, O4, C6 atoms of Neu5using atleast-squares approach. The superposition aligns the C1 carboxylate, C3hydroxyl, and C4 acetylamino groups of HNBA with the C2 carboxylate, O4hydroxyl, and C4 acetylamino groups of Neu5Ac2en and preserves theimportant interactions of these groups with the bacterial sialidaseactive site residues in the HNBA-bacterial sialidase modeled complex.See FIG. 3a.

The GBA-bacterial sialidase complex was modeled by a least squaressuperposition of the C1, N3, and C5 atoms of GBA onto the C2, C7, and C3atoms of Neu5Ac2en . See FIG. 3b. The superposition aligns the GBAguanidinium group to the Neu5Ac2en glycerol group in the bacterialsialidase active site. This orientation corresponds to the binding modeobserved in the GBA influenza virus type A N2 sialidase complex.Following the initial least squares superposition, the guanidiniumsidegroup of GBA was manually rotated into the N3⁺ binding site Aidentified in the GRID map analysis on a graphics display using theprogram FRODO.

All of the water molecules identified in the Neu5Ac2en -bacterialsialidase crystal structure were included in the HNBA-bacterialsialidase model. The criteria for retaining the waters in theHNBA-bacterial sialidase complex was that none of the water moleculeswere sterically excluded by the presence of the HNBA inhibitor and allof the water molecules possessed potential hydrogen bonding partners. Inthe GBA-bacterial sialidase, two of the water molecules in the Neu5Ac2en-bacterial complex, HOH 906 and HOH 907, were excluded due to stericoverlap with the GBA guanidino group.

The HNBA and GBA bacterial sialidase complexes were energy minimizedusing a conjugate gradient protocol within the program X-PLOR to relievesteric conflicts that may have resulted from the Neu5Ac2ensuperposition. A harmonic constraint of 500 kcal/mol was placed on atomsmore than 10 Å distant from the benzoic acid compound, while those atomswithin a 10 Å radius of the benzoic acid compound had no harmonicconstraints. The active site geometry in the energy minimized benzoicacid-bacterial sialidase complexes was almost identical to that observedin the energy minimized Neu5Ac2en -bacterial sialidase complex. Energyminimization of the HNBA-bacterial sialidase complex did notsignificantly alter the orientation of HNBA in the bacterial sialidaseactive site. Surprisingly, energy minimization of the GBA-bacterialsialidase complex changed the orientation of GBA in the bacterialsialidase active site when compared to the starting position (Neu5Ac2enleast squares superposition). In the energy minimized GBA-bacterialsialidase complex, the benzene ring of GBA is rotated approximately 20°around the inhibitor carboxylate-acetylamino axis. The rotation placesthe GBA guanidino group closer to the N3⁺ binding site and tilts thebenzene ring C5 and C6 atoms away from the active site floor. Despitethe tilt in the GBA benzene ring, no change in orientation was observedfor the GBA carboxylate and N-acetylamino groups in the energy minimizedGBA-bacterial sialidase complex when compared to the energy minimizedHNBA-bacterial sialidase complex. In addition, the active site residuesin both of the energy minimized benzoic acid-bacterial sialidasecomplexes adopt conformations which are analogous to the active siteresidues of the energy minimized Neu5Ac2en -bacterial sialidase. Theroot-mean-square (rms) deviation for sialidase atoms within the 10 Åradius between the energy minimized Neu5Ac2en and HNBA-bacterialsialidase complexes, is 0.05 Å, between the minimized Neu5Ac2en andGBA-bacterial sialidase complexes, 0.07 Å.

As used herein, the term "salt" refers to the cation, such as Li⁺, K⁺,Na⁺, Ca²⁺, Mg²⁺, Al³⁺ etc., which corresponds to the COO⁻ group of theinhibitors. Sodium salts are often preferable for pharmaceuticalcompositions. One of ordinary skill in the art would recognize that thefundamental utility of the compounds is not dependent upon the identityof the particular cation. As shown, H⁺ is also a suitable cation.

The program DELPHI calculates the electrostatic potential ofmacromolecular systems using a finite difference solution to thenon-linear Poisson-Boltzmann equation [Gilson, M K, & Honig, B,Calculation of the total electrostatic energy of a macromolecularsystem; solvation energies, binding energies, and conformationalanalysis. Proteins: structure, function and genetics, 4, pp. 7-18(1988)]. For a given macromolecular inhibitor-protein complex, DELPHIcan be used to calculate the total electrostatic energy of the system.For the modeled complexes, the electrostatic contribution to the freeenergy change upon complex formation, ΔG_(el), was derived from thetotal electrostatic energies of three inhibitor complexes: E1, theelectrostatic energy of the complex when charges are present only on theprotein residues; E2, the electrostatic energy of the complex whencharges are present only on the inhibitor residues; and E3, theelectrostatic energy of the complex when charges are present on both theprotein and inhibitor residues. The free energy change resulting fromelectrostatic interactions is therefore: ΔG_(el) =E₁ -(E₂ +E₃). Becausethe binding of inhibitors to the bacterial active site is dominated byelectrostatic interactions and due the inherent complexity ofhydrophobic interactions, the contribution of the hydrophobic effect wasnot explicitly included in the DELPHI calculation. However, thecontribution of the hydrophobic effect to the free energy of complexformation for compounds within a single class, which have a similarfunctional groups and chemical properties, is roughly on the same orderof magnitude. Therefore, exclusion of the hydrophobic contribution tothe calculated free energy of complex formation should not change therelative ranking of a series of compounds within any single class.

For each of the modeled complexes, the electrostatic contribution to thechange in free energy of complex formation was calculated using aprotein/inhibitor dielectric constant of 4, a solvent dielectricconstant of 80, an ionic strength of 0.145 M, and a focusing protocol of30→90% fill. Table 7 presents the calculated free energies of complexformation for the sialidase complexes, as well as, the calculated freeenergies of complex formation for the benzoic acid-bacterial sialidasecomplexes.

                  TABLE 7                                                         ______________________________________                                        DELPHI electrostatic energies of complex formation for HNBA and GBA            when complexed to bacterial sialidase from Salmonella typhimurium.                                                  ΔG.sub.el                                                                     Δ(ΔG)†                                                            E.sub.1                                                                E.sub.2       E.sub.3                                                         ΔG.sub.el     (kcal/                                                                (kcal/                 Data Set   (kT)          (kT)          (kT)         (kT)           mol)                                                                  mol)             ______________________________________                                        GBA    29341.89  99.49  29388.96                                                                             -52.42                                                                              -31.09                                                                              -12.87                               HNBA       29438.42      107.24       29514.95       -30.71    -18.21                                                          0.00                       ______________________________________                                         \Δ(ΔG) = ΔG.sub.el (i) - ΔG.sub.el          (HNBA), where i is any inhibitor.                                        

The partial charges assigned to the compounds in the DELPHI calculationwere determined using the semiemperical program MOPAC v6.0 and thecoordinates of the final energy minimized inhibitor compound whencomplexed to S. typhimurium. The DELPHI electrostatic energies werecalculated using a focusing protocol comprised of three stages of thepercent fill: 30%, 60%, and 90%. Using 90% fill in the final stage gavea step size of 1.04 Å. The following parameters were used for all DELPHIcalculations: protein dielectric, e_(p) =4, solvent dielectric, e_(s)=80, ionic strength=0.145, linear iterations=1000, and non-lineariterations =3000. Unless otherwise stated, the DELPHI default valueswere used for all other parameters in the electrostatic free energycalculations.

L. Comparison of Influenza Virus and Bacterial Sialidase Active Sites

As with influenza virus sialidases, the bacterial sialidase active sitefrom S. typhimurium also contains an arginine triad (Arg 37, Arg 246,and Arg 309), which binds the carboxylate moiety of sialic acid; ahydrophobic pocket, which accommodates the methyl group of the substrateN-acetylamino moiety; and a tyrosine residue (Tyr 342) located beneaththe substrate pocket. Though functionally similar, the residues whichcompose the hydrophobic pocket differ between the bacterial and viralactive sites. The bacterial hydrophobic binding site is composed of twotryptophans (Trp 121, Trp 128), one methionine (Met 99), and one leucine(Leu 175), where as the viral hydrophobic pocket contains just onetyrosine (which is analogous in position to Trp 128 of S. typhimurium),one isoleucine and one arginine sidechain. In addition, several otherfeatures can be used to distinguish the S. typhimurium bacterial activesite from its counterpart in the influenza virus sialidase. For example,there are several significant differences in the active site residueswhich form the binding pockets for the O4 hydroxyl and glycerol groupsof sialic acid. In the bacterial enzyme, an arginine and an asparticacid residue (Arg 56 and Asp 100 in S. typhimurium ), which the viralsialidase does not contain, form strong hydrogen bonds to the O4 atom ofthe bound sialic acid. The presence of these residues in the bacterialactive site also prevent binding of sialic acid analogs modified at theO4 position with large, bulky groups. At the glycerol binding pocket,the bacterial enzyme is lacking a glutamic residue (Glu 275) found inthe influenza virus enzyme which provides two hydrogen bonds to theglycerol O8 and O9 atoms of the bound sialic acid. In addition, in theglycerol pocket found in the bacterial sialidase is much larger andbroader than the glycerol pocket of influenza virus sialidase due to thealternate orientation of a loop in the bacterial sialidase. In thebacterial enzyme, the loop, comprised of residues 196-205, points awayfrom the active site to create a wide, shallow glycerol binding pocket.The analogous loop in the influenza virus sialidase points toward theactive site and effectively limits the size of the pocket to inhibitorsidegroups no longer than glycerol.

M. Biological Significance

Bacterial and trypanosomal infections of humans and livestock can resultin serious medical complications and economic loss. Though anti-bioticsare available for the treatment of bacterial infections, inhibitors ofbacterial sialidase may be medically useful where sialidase activity hasbeen correlated with severe bacterial infection pathology.

N. Modes of Administration

As used herein, the effective amount of a compound of the inventionrequired for use in the methods described herein will vary not only withthe particular compound selected but also with the mode ofadministration, the nature of the condition in the subject, and the ageand health of the subject. The exact dosage will ultimately bedetermined by a physician or other person skilled in the art. However, asuitable systemic dose will generally range from about 0.01 to about 200mg/kg of bodyweight per day. More preferably, an effective amount(suitable dose) will range from 0.1 to 50 mg/kg/day. Treatment may occurbefore bacterial infection (i.e. prophylaxis), at the start ofinfection, or after the onset of established symptoms or infection.Treatment with the effective amount may be given 1 to 4 times daily andthe typical duration will range from 3 to 10 days, or until bacteria ortrypanosoma are no longer present and/or symptoms have disappeared.Furthermore, a suitable topical dose will generally range fromapproximately 1 nM to 1 mM. Treatment may occur before bacterialinfection (.e. prophylaxis), at the start of infection, or after theonset of established symptoms or infection. Treatment with the effectiveamount may be given 1 to 4 times daily and the typical duration willrange from 3 to 10 days, or until bacteria or trypanosoma are no longerpresent and/or symptoms have disappeared. Those skilled in the art willrecognize that deviations from the above described treatment methods andeffective amounts are possible and are to be included in the subjectmatter taught herein.

Furthermore, it is possible that, during therapy, the compounds may beadministered alone as pure chemical or as a pure pharmaceuticallyacceptable salt, analog or derivative. However, it is preferable toprovide the active chemical, or its pharmaceutically acceptable salt,analog or derivative, as a pharmaceutical formulation, either as a drypowder (tablet or capsule form or with a suitable carrier), or as asolution or suspension (in water or in physiologically acceptablesolvents or cosolvents such as ethanol, propylene glycol, or PEG 400).The appropriate pharmaceutical formulation may be administered bytopical, oral, intranasal, intravenous, intramuscular or otherappropriate modes. A preferred mode of administration for treating abacterial infection is to topically apply the inhibitor to the infectedregion of the subject. The desired dosage (effective amount) may beadministered in one or in divided doses at appropriate intervals eachday. The compounds and compositions of the invention may also beadministered in combination with other therapeutic agents. Those skilledin the art will appreciate that dosages and modes of administration arereadily determinable without undue experimentation.

The compounds of the invention may be conveniently formulated intopharmaceutical compositions composed of one or more of the compounds inassociation with a pharmaceutically acceptable carrier. See, e.g.,Remington's Pharmaceutical Sciences, latest edition, by E. W. MartinMack Pub. Co., Easton, Pa., which discloses typical carriers andconventional methods of preparing pharmaceutical compositions that maybe used in conjunction with the preparation of formulations of theinventive compounds and which is incorporated by reference herein.

In preferred embodiments, the compounds of the present invention can bedelivered in the following manners:

1. Oral/Pill

2. Nasal Aerosol

3. Ear/Eye Drops

4. Topical Cream

5. Intravenous Solution

6. Suppository

7. Dental Mouthwash

These methods of drug delivery may be practiced with standardpharmacological formulations.

O. Conditions Treated or Prevented

The compounds of the invention provide a broad range of anti-bacterialand anti-trypanosomal activity. The compounds can inhibit bacterialsialidase and trypanosomal trans-sialidase. The medical benefits whichresult from inhibition of sialidase/trans-sialidase activity may vary asthey are dependent on the specific organism. Examples wheresialidase/trans-sialidase activity has been documented to play a role inthe pathology are listed below. One skilled in the art would recognizethat treatment with the compounds to inhibit thesialidase/trans-sialidase activity in these cases would provebeneficial.

The present compounds can be used in methods of preventing bacterial ortrypanosome adherence. In the following examples, the organism requiresthe sialidase/trans-sialidase activity to attach to the cells of thehost prior to invasion. The treatment with compounds would therefore beexpected to prevent or limit infection of the host by the microorganism,but not directly kill the microorganism. Treatments, or prophylaxis, ofthe following infections can be effective:

a) dental caries/bacterial-mediated gum disease [Childs, W. d. &Gibbons, R. J. (1990). Selective modulation of bacterial attachment tooral epithelial cells by enzyme activities associated with poor oralhygiene. J Period Res., 25, pp. 172-8.; Liljemark, W. F., Bloomquist, C.G., Fenner, L. J., Antonelli, P. J. & Coulter, M. C. (1989). Effect ofneuraminidase on the adherence to salivary pefficle of Streptococcussanguis and Streptococcus mitis. Caries Res., 23, pp. 141-5; Rogers, R.,Newbrun, E. & Tatevossian, A. (1979). Neuraminidase activity in humandental plaque fluid. Archives of Oral Biology 24(9), 703-5]. Mode ofdelivery: dental mouthwash;

b) arteritis [Nakato, H., Shinomiya, K. & Mikawa, H. (1986). Possiblerole of neuraminidase in the pathogenesis of arteritis andthromboctopenia induced in rats by Erysipelothrix rhusiopathiae. PatholRes Pract., 181, pp. 311-9]. Modes of delivery: oral pill, intravenoussolution;

c) Pseudomonas aeruginosa infection in cystic fibrosis (CF) [Cacalano,G., Kays, M., Saiman, L. & Prince, A. (1992). Production of thePseudomonas aeruginosa neuraminidase is increased under hyperosmolarconditions and is regulated by genes involved in alginate expression.Journal of Clinical Investigation 89(6), 1866-74]. Modes of delivery:oral pill, intravenous solution, nasal aerosol;

d) Actinomyces viscosus and A. naeslundii infection [Costello, A H,Cisar, J, Kolenbrander, P E & Gabriel, O (1979). Neuraminidase-dependenthemagglutination of human erythrocytes by human strains of Actinomycesviscosus and Actinomyces naeslundii. Infection & Immunity 26(2),563-72]. Modes of delivery: oral pill, intravenous solution, topicalcream;

e) Bacteroides fragilis infection [Guzman, C. A., Plate, M. & Pruzzo, C.(1990). Role of neuraminidase-dependent adherence in Bacteroidesfragilis attachment to human epithelial cells. Fems Microbio Lett., 59,pp. 187-92; Namavar, F., Van der Bijl, M. W., Appelmelk, B. J., DeGraaff, J. & MacLaren, D. M. (1994). The role of neuraminidase inhaemagglutination and adherence to colon WiDr cells by Bacteroidesfragilis. J Med Microbiol., 40, pp. 393-6]. Modes of delivery: oralpill, intravenous solution, topical cream; and

f) Chagas' Disease, Tiypanosoma cruzi infection [de Titto & Araujo,1987;

Ming et al., 1993; Prioli et al., 1991]. Modes of delivery: oral pill,intravenous solution.

Furthermore, the inhibitors can be used in the prevention of bacterialvaginosis. In this example, sialidase is highly correlated with theprogress of the disease. The most probable role of sialidase is forsuccessful attachment and colonization of the upper and lower genitaltract. Therefore, treatment with the compounds would be expectedprimarily to prevent or slow the progress of bacterial infection toallow the host's immune system time to recover. A second, but importantresult of treatment with the compounds of the invention would be toreduce the symptoms associated with bacterial vaginosis (rash, itching,discharge, etc.) [Briselden, A. M., Moncla, B. J., Stevens, C. E. &Hillier, S. L. (1992). Sialidases (neuraminidases) in bacterialvaginosis and bacterial vaginosis-associated microflora. J ClinMicrobiol., 30, pp. 663-6; McGregor, J. A., et al. (1994). Bacterialvaginosis is associated with prematurity and vaginal fluid mucinase andsialidase: results of a controlled trial of topical clindamycin cream.Am J Obstet Gynecol., 170, pp. 1048-59]. Modes of delivery: topicalcream, suppository, oral pill.

Also, the present inhibitors can be used for the prevention of inner eareffusion. Sialidase activity has been correlated with the development ofacute and chronic otitis in inner ear effusions. Treatment with thecompounds would therefore prevent the damage to the inner ear mucosa andprevent otitis from developing. It would not directly kill the organismcausing the infection [LaMarco, K. L., Diven, W. F. & Glew, R. H.(1986). Experimental alteration of chinchilla middle ear mucosae bybacterial neuraminidase. Ann Otol Rhinol Laryngol., 95, pp. 304-8;LaMarco, K. L., Diven, W. F., Glew, R. H., Doyle, W. J. & Cantekin, E.I. (1984). Neuraminidase activity in middle ear effulsions. Annals ofOtology, Rhinology & Laryngology 93(1 Pt 1), 76-84]. Modes of delivery:Ear drops, oral pill

Prevention of arthritis symptoms. Sialidase activity has been correlatedwith the disease severity in arthritic rats. The effect of treatmentwith the compounds may reduce the symptoms associated with arthritis,such as lymphocyte activation and swelling [Marchand, N. W., Kishore, G.S. & Carubelli, R. (1978). Neuraminidase activity in the blood and liverof arthritic rats. Experimental & Molecular Pathology 19(3), 273-80].Modes of delivery: oral pill, intravenous solution, topical cream.

Prevention of hemolytic uremic syndrome (HUS) in patients with pneumoniaand hemolytic anemia. Sialidases have been implicated as the agent whichexposes the Thomsen cryptantigen. Treatment with the compounds wouldreduce the prevalence of patients developing hemolytic uremic syndrome,but not cure the underlying causes [Seger, R., Joller, P., Baerlocher,K., Kenny, A., Dulake, C., Leumann, E., Spierig, M. & Hitzig, W. H.(1980). Hemolytic-uremic syndrome associated withneuraminidase-producing microorganisms: treatment by exchangetransfusion. Helvetica Paediatrica Acta 35(4), 359-67]. Modes ofdelivery: oral pill, intravenous solution

Prevention of group B streptococci infection in neonates. The highlevels of sialidase activity have been associated with severe group Bstreptococci infection in infants, where streptococci infection can leadto diarrhea, weight loss, or more severe complications. Treatment withthe compounds would prevent bacterial spread and reduce the symptomsassociated with the disease in infants [Miffigan, T. W., Baker, C. J.,Straus, D. C. & Mattingly, S. J. (1978). Association of elevated levelsof extracellular neuraminidase with clinical isolates of type imi groupB streptococci. Infection & Immunity 21(3), 738-46]. Modes of delivery:intravenous solution, suppository

Prevention of acute poststreptococcal glomerulonephritis. The sialidaseactivity of virulent streptococcal infections has been shown to play arole in the development of acute poststreptococcal glomerulonephritis.It would therefore follow that compound treatment would decrease thelikelihood of developing acute poststreptococcal glomerulonephritis[Mosquera, J. & Rodriguez-Iturbe, B. (1984). Extracellular neuraminidaseproduction of streptococci associated with acute nephritis. ClinNephrol., 21, pp. 21-8; Mosquera, J. A., Katiyar, V. N., Coello, J. &Rodriguez-Iturbe, B. (1985). Neuraminidase production by streptococcifrom patients with glomerulonephritis. Journal of Infectious Diseases151(2), 259-63; Potter, E. V., Shaughnessy, M. A., Poon-King, T. &Earle, D. P. (1982). Streptococcal neuraminidase and acuteglomerulonephritis. Infection & Immunity 38(3), 1196-1202]. Modes ofdelivery: intravenous solution, oral pill

Prevention of acne and seborrheic eczema. Sialidase activity has beenhighly associated with Propionibacterium acnes-strains isolated frompatients with acne vulgaris, seborrheic eczema and healthy subjects.Treatment with the compounds of the invention should therefore preventor decrease infection by the Propioniobacterium acnes bacterium. Itshould also alleviate some of the symptoms associated with acne[Hoffier, U., Gloor, M. & von Nicolai, H. (1981). Neuraminidaseproduction by Propionibacterium acnes-strains isolated from patientswith acne vulgaris, seborrheic eczema and healthy subjects. ZentralblattFur Bakteriologie, Mikrobiologie Und Hygiene 250(1-2), 122-6; vonNicolai, H., Hoffler, U. & Zilliken, F. (1980). Isolation, purification,and properties of neuraninidase from Propionibacterium acnes.Zentralblait Fur Bakteriologie 247(1), 84-94]. Modes of delivery:topical cream, oral pill

Prevention of arteritis. Erysipelothrix rhusiopathiae induced arteritiswas highly correlated to production of sialidase by the bacteria.Treatment would therefore inhibit bacterial sialidase, attachment, andinfection of aortic tissue. [Nakato et al, 1986; Nakato, H., Shinomiya,K. & Mikawa, H. (1987). Adhesion of Erysipelothrix rhusiopathiae tocultured rat aortic endothelial cells. Role of bacterial neuraminidasein the induction of arteritis. Pathology, Research & Practice 182(2),255-60]. Modes of delivery: oral pill, intravenous solution

This invention thus describes classes of bacterial sialidase inhibitors,their pharmaceutically acceptable salts and derivatives, and mixturesthereof having general structure I. These inhibitors may be used in avariety of methods as described herein.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A method of inhibiting bacterial sialidasecomprising administering to a subject an inhibiting effective amount ofa compound of formula I: ##STR23## wherein A is CO₂ H, PO₂ H, or SO₂ H;B is N; R₁ and R₂ are H; R₃ and R₄ are, independently, H, OH, NO₂,guanidino, or alkyl or alkenyl of from 1 to 3 carbons where the alkyl oralkenyl is unsubstituted or is substituted, independently, with one ormore of OH, NH2, or halide; R₅ is H; and R₆ is COCH₃, or COCI₃ ; or ananalog, pharmaceutically acceptable salt, derivative, or mixturethereof.
 2. The method of claim 1, wherein A is CO₂ H; R₃ and R₄ are,independently, H, OH, NO₂, or guanidino; R₅ is H; and R₆ is COCH₃, or ananalog, pharmaceutically acceptable salt, derivative, or mixturethereof.
 3. The method of claim 2, wherein one of R₃ and R₄ is OH theother is NO₂.
 4. The method of claim 2, wherein one of R₃ and R₄ is Hthe other is guanidino.
 5. A method of treating a bacterial ortrypanosomal infection, comprising administering to a subject apreventative effective amount a compound of formula I: ##STR24## whereinA is CO₂ H, PO₂ H, or SO₂ H; B is N; R₁ and R₂ are H; R₃ and R₄ are,independently, H, OH, NO₂, guanidino, or alkyl or alkenyl of from 1 to 3carbons where the alkyl or alkenyl is unsubstituted or is substituted,independently, with one or more of OH, NH2, or halide; R₅ is H, and R₆is COCH₃, or COCI₃ ; or an analog, pharmaceutically acceptable salt,derivative, or mixture thereof.
 6. The method of claim 5, wherein A isCO₂ H; R₃ and R₄ are, independently, H, OH, NO₂, or guanidino; R₅ is H;and R₆ is COCH₃, or an analog, pharmaceutically acceptable salt,derivative, or mixture thereof.
 7. A method of preventing a bacterial ortrypanosomal infection, comprising administering to a subject apreventative effective amount a compound of formula I: ##STR25## whereinA is CO₂ H, PO₂ H, or SO₂ H; B is N; R₁ and R₂ are H; R₃ and R₄ are,independently, H, OH, NO₂, guanidino, or alkyl or alkenyl of from 1 to 3carbons where the alkyl or alkenyl is unsubstituted or is substituted,independently, with one or more of OH, NH2, or halide; R₅ is H; and R₆is COCH₃, or COCI₃ ; or an analog, pharmaceutically acceptable salt,derivative, or mixture thereof.
 8. The method of claim 7, wherein A isCO₂ H; R₃ and R₄ are, independently, H, OH, NO₂, or guanidino; R₅ is H;and R₆ is COCH₃, or an analog, pharmaceutically acceptable salt,derivative, or mixture thereof.
 9. The method of claim 1, wherein theadministering step comprises topical administration.
 10. The method ofclaim 5, wherein the administering step comprises topicaladministration.
 11. The method of claim 7, wherein the administeringstep comprises topical administration.